Being able to control and manipulate gene function and gene expression forms the basis of recombinant DNA engineering. For example, over time, various methods have been developed for knocking out gene expression. By knocking out a gene's activity, it is possible to study its function.
Traditionally, a direct gene disruption method (i.e., also later referred to as “the conventional knockout method”) was used to knock out a gene. This involved inserting a DNA fragment containing a desired mutant gene (or part of a gene) into a vector and then introducing the vector into, for example, embryo-derived mouse stem cells (i.e., embryonic stem cells or ES cells). The DNA fragment could include, for example, a neomycin selectable marker. Most insertions would occur in random sites in the mouse genome. So long as the fragment introduced itself into an exon upon introduction into an ES cell, the fragment would disrupt the expression of the gene of which the exon is a part. However, by this method, there was no control over when a gene might be knocked out. Also, if a gene were developmentally required, even if for a role that was different than the one that a scientist was interested in studying, then there was nothing that the scientist could do —the gene could end up causing developmental lethality, thus making it impossible to further pursue a study of the gene's later role.
The conditional knockout method based on recombinase mediated deletion was an improvement over the conventional knockout method. Using the conditional knockout method, a scientist would introduce, for example, Lox sites (i.e., short for “locus of crossover” sites) into the introns of a gene that is to be disrupted. Lox sites are DNA sequences that, when found in a pair in the same orientation in a length of DNA, function to remove any intervening DNA sequence found between the two LoxP sites when in the presence of Cre recombinase (i.e., or “Cre”), a 38 kD site-specific recombinase. (Cre is short for causes recombination.) As shown in FIG. 1, using a conditional knockout strategy, since the LoxP sites are to be inserted into the introns of genes, they remain inert, in theory, and do not affect the mature transcript that ends up being translated into a protein when Cre is absent. This made the knocking out of the gene into which the Lox sites have been inserted conditional upon the delivery of Cre to the cell. Cre could then be delivered using a vector, such as a viral vector.
The deletion-based conditional knockout method is illustrated in FIG. 1. In FIG. 1, the first allele shows two Lox sites as triangular elements in the same orientation inserted in two intron sequences of a gene that has three exons. The Lox sites flank two exons. The arrow at the first exon shows where transcription starts for the gene. In the absence of Cre, the gene in the first allele (i.e., all its exons) should get transcribed and translated normally. However, upon the addition of Cre, recombination between LoxP sites causes the second and third exons to be removed, as shown in the second allele. As a result, the gene in the second allele is disrupted, i.e., knocked out.
Deletion-based conditional knockout animal models are advantageous over direct gene disruption animal models in that the former avoids developmental lethality. For example, if a gene is initially important for liver development in an organism but later is expressed in the organism's heart tissue, and it is the gene's expression in heart tissue that a scientist wishes to study, the scientist can use a conditional knockout strategy to keep the gene intact during development, and then to disrupt the gene in heart tissue upon specific delivery of Cre to that tissue. This phenomenon also leads to greater control and flexibility in experimentation, since one can conduct a temporal and/or tissue specific knockout based on the mechanism of Cre delivery.
However, a disadvantage of the deletion-based conditional knockout strategy involving the Cre/LoxP system is that the scientist must know a great deal about the gene under study, including its essential domains, so as to be able to properly insert the Lox sites. If the Lox sites end up disrupting an exon, they will not be inert. Also, it is important that the Lox sites flank important parts of a gene that are critical to gene function. Moreover, the scientist must be aware of all the different transcripts that might be encoded by the same gene. As well, when genes are very large or have multiple essential exons that are far apart from one another, it may be very difficult to engineer a conditional knockout mutant gene. Thus the construct design and preparation for conditional knockout experiments are more complicated than in conventional knockout by gene disruption experiments.
Yet another method exists for knocking out genes called gene trapping, as shown in FIG. 2. The gene trap method provides an approach for creating random gene knockouts in cells and animals as opposed to the gene specific approach of gene targeted knockouts provided, for example, by the two methods described above. The first allele shown on FIG. 2 is a wild-type gene with three exons, and has a transcription start site at the arrow. The second allele in FIG. 2 illustrates a gene trap, a single genetic construct that is introduced into an intron. The gene trap would also function if it were inserted into an exon. For example, the gene trap shown in FIG. 2 contains a splice acceptor (“SA”) that forces splicing from any exon to itself during transcription, a reporter gene (i.e., LacZ) that, because of the SA, will get transcribed as a hybrid message with the initial portion of the wild-type gene, and a neomycin resistance gene which is used to select any cells that might have incorporated the gene trap into its genome. Gene traps cause random gene disruption since they are inserted randomly into the genome. As a result, they can be used for the in vitro identification and discovery of new genes. In this regard, the gene trap provides a genetic tag that can be used to identify the trapped gene. However, gene trapping does not provide much experimental control and does not allow a scientist, for example, to track a particular gene from development and then disrupt the specific gene at a particular point in time.
Therefore, in summary, the conventional knockout method involves a simple construction and is gene specific, the deletion-based conditional knockout method involves a complex construction and is gene specific, and the gene trapping knockout method is simple and random.